Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene disruption,
Gene mutation,
Introduction of a transgene
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Reference (PubMed-PMID number) |
Not published (yet)
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MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone |
P. berghei ANKA 676m1cl1 (RMgm-29)
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Other information parent line | 676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
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The mutant parasite was generated by |
Name PI/Researcher | Costa DM, Tavares J |
Name Group/Department | IBMC – Instituto de Biologia Molecular e Celular |
Name Institute | Universidade do Porto |
City | Porto |
Country | Portugal |
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Name of the mutant parasite |
RMgm number | RMgm-5184 |
Principal name | SPATR KD (knock-down) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Neither the number nor the size of the oocysts revealed any clear developmental impairments. However, a significant increase in the number of sporozoites per oocyst was observed in the SPATR KD line on day 15, indicating a defect in egress of sporozoites from the oocyst. |
Sporozoite | SPATR KD-infected mosquitoes showed strongly reduced sporozoite numbers. The reduced numbers of sporozoites in the salivary glands did not result in the accumulation of sporozoites in the hemolymph. Few SPATR KD sporozoites were detected within the salivary glands and these did not show obvious structural modifications by transmission electron microscopy analysis. |
Liver stage | To evaluate sporozoite infectivity, sporozoites were collected from the hemolymph and injected intravenously in mice. While WT parasites successfully accomplished the liver stage and subsequent blood infections, parasite liver loads in SPATR KD-infected mice were strongly reduced and were rarely able to establish a blood infection. A milder defective phenotype was observed when we infected mice with the few SPATR KD sporozoites that we were able to collect from salivary glands, with reduced parasite liver loads and prolonged prepatent periods of approximately 1 day compared to WT.
Evidence is presented that SPATR KD sporozoites exhibit defects in hepatocyte invasion, migration and homing to the liver. |
Additional remarks phenotype | Mutant/mutation
In this 'promoter-swap' mutant, first an additional copy of the spatr gene (under control of the promoter of PBANKA_0603900 (hado; haloacid dehydrogenase domain ookinete protein)) has been introduced into the silent intergenic locus in chromosome 6 (SIL6) and subsequently the endogenous spatr gene has been deleted. The mutant expresses GFP-Luc under control of the eef1a promoter.
Published in bioRxiv preprint doi: https://doi.org/10.1101/2022.03.06.483110
Protein (function)
The secreted protein with an altered thrombospondin repeat (SPATR) contains two adhesive domains: an atypical thrombospondin type-1 repeat (TSR) and a Type II epidermal growth factor (EGF)-like domain. It is conserved across Plasmodium species and has several orthologues among the Apicomplexa phylum. Although its gene is transcribed in all invasive stages (sporozoites, merozoites and ookinetes), SPATR levels are upregulated in mature sporozoites of both human and rodent infecting Plasmodium species. Moreover, immunolocalization studies detected the protein at the surface of sporozoites of several Plasmodium species, despite the lack of predicted transmembrane domains or a glycosylphosphatidylinositol anchor. Antibodies against the SPATR of P. falciparum also blocked hepatocyte invasion by sporozoites.
Phenotype
Normal asexual blood stage development, gametocyte, ookinete and oocyst production.
Neither the number nor the size of the oocysts revealed any clear developmental impairments. However, a significant increase in the number of sporozoites per oocyst was observed in the SPATR KD line on day 15, indicating a defect in egress of sporozoites from the oocyst.
SPATR KD-infected mosquitoes showed strongly reduced sporozoite numbers. The reduced numbers of sporozoites in the salivary glands did not result in the accumulation of sporozoites in the hemolymph. Few SPATR KD sporozoites were detected within the salivary glands and these did not show obvious structural modifications by transmission electron microscopy analysis. To evaluate sporozoite infectivity, sporozoites were collected from the hemolymph and injected intravenously in mice. While WT parasites successfully accomplished the liver stage and subsequent blood infections, parasite liver loads in SPATR KD-infected mice were strongly reduced and were rarely able to establish a blood infection. A milder defective phenotype was observed when we infected mice with the few SPATR KD sporozoites that we were able to collect from salivary glands, with reduced parasite liver loads and prolonged prepatent periods of approximately 1 day compared to WT.
Evidence is presented that SPATR KD sporozoites exhibit defects in hepatocyte invasion, migration and homing to the liver.
Additional information
SPATR is expressed in blood stages, ookinetes and in sporozoites and has an essential function in blood stages. To achieve downregulation of expression in sporozoites while maintaining blood stage expression, a promoter swap approach was used to generate a mutant where the Plasmodium berghei spatr gene was placed under transcriptional control of the hado gene promoter.
Available RNA-seq datasets showed higher abundance of spatr transcripts in blood schizonts, ookinetes and salivary gland sporozoites. Based on these criteria, we searched genome-wide RNA-seq data and selected the genes that encode the haloacid dehydrogenase domain ookinete protein (HADO).
Transcripts were found to be significantly less abundant in SPATR KD oocysts and sporozoites from days 11–12 post-infection than in the control lines. Protein levels were drastically reduced in SPATR KD hemolymph sporozoites.
Other mutants |